DNA extraction from blood.
Part A: Purifying nuclear pellets.
1) Add 50-60ul fresh, packed red blood cells (RBC) to 700ul PBS. Mix by inversion.
2) Add 700-800ul Lysis buffer. Close tube. Vortex briefly (2-5s)
3) Centrifuge for 15s at 12,000 rpm. Remove red supernatant by aspiration.
4) Add 1.0ml new Lysis buffer. Vortex (15-30s) to break up pellet.
6) Centrifuge for 15s at 12,000 rpm. Remove red supernatant by aspiration.
** at this stage nuclear pellets may be stored long term by adding 100ul TEN buffer and freezing at 20c.
Part B: Extraction of DNA from nuclei.
1) Transfer pellets to 50ml tubes to digest by either:
a) Adding 1ml TEN buffer to pellet in microfuge tube. Vortex well (>30s) to break up pellet. Pour contents into empty 50ml tube. Add 5ml TEN w/ 0.5%SDS. OR
b) Use loop to move pellet into empty 50 ml tube. Add 6ml TEN w/ 0.5%SDS.
2) Add 120ul proteinase-K. Swirl to mix.
3) Incubate tubes at 55c for 12 hrs to 3 days with occasional vigorous mixing.
**) For steps 8-11 wear safety glasses and gloves. Work in hood.
4) Remove tubes from oven and carefully add 5ml PCI (phenol: chloroform: IAA)
5) Closed tubes tightly. Vortex briefly (5s) to mix.
6) Spin tubes in balanced large centrifuge for 10-15min at 6,000 rpm.
7) Carefully remove 5-6ml aqueous layer for each sample into new, clean microfuge tube. Be sure to avoid transferring any of the interface or PCI layers. Pour remaining PCI liquid portion into liquid hazardous waste and tube into bag in hood.
8) Add 1/3 volume (1.5-2ml) 10M (NH4)AC to each tube. Close top. Mix.
9) Add 15-20ml 100% Ethanol. Close top.
10) Invert and swirl to mix and precipitate DNA.
-If no visible large clot, let sit in -20c freezer for ½ - 2 days.
11) Hook out large clots with plastic loop and transfer to microfuge tube pre-filled with 1.0ml 70-80% Ethanol. Let sit at 4c for 20min to 2 days.
12) Spin pellets at 12,000 rpm for 3-5min. Remove Ethanol wash by aspiration.
13) Let pellet dry 5-30min at RT. Add 200-500ul TE soln and let resuspend at 4c.
14) Check DNA concentration on flourometer and quality on gel. Adjust as needed.